This research project seeks to elucidate the enzyme mechanisms involved in the assimilatory reduction of nitrate to ammonia. The significance of this process is evident since the various oxidized inorganic forms of nitrogen provide the ultimate nitrogen source for all life. Nitrate assimilation is a two-step process: the 2e minus reduction of nitrate to nitrite, followed by the 6e minus reduction of NO minus over 2 to ammonia. The enzymes catalyzing these reactions, nitrate reductase and nitrite reductase, are adaptively formed in Neurospora crassa in the presence of either NO minus over 3 or NO minus over 2. Their synthesis is repressed by NH plus 4. The nitrate reductase transfers electrons from NADPH to NO minus 3 via its component functional groups according to the following scheme: NADPH yields ((-SH) yields FAD yields cytochrome b557 yields Mo) yields NO minus over 3. The role of essential sulfhydryls as the primary acceptor of reducing equivalents was indicated by recent studies using diazotized aminopyridine adenine dinucleotide as an NADPH site-specific reagent. The nitrite reductase catalyzes the stoichiometric reduction of NO minus over 2 to NH plus 4 using 3 equivalents of reduced pyridine nucleotide via the sequence: NAD(P)H yields ((FAD,-SH) yields siroheme) yields NO minus over 2. The nitrite reductase also displays FAD-dependent NAD(P)H-diaphorase activity and flavin-independent, dithionite-nitrite reductase activity. This laboratory has purified the N. crassa nitrite reductase (mol. wt. 300,000) to homogeneity. Primary objectives at this time involve the determination of the subunit organization of the enzyme complex, the nature of the various electron transport functions, and the relationships between the structure and the function of the nitrite reductase. BIBLIOGRAPHIC REFERENCES: Amy, N.K., R.H. Garrett and B.M. Anderson, "Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs." Biochemica et Biophysica Acta, 1976 (in press). Garrett, R.H., "Nitrite Reduction in Fungi", Microbiology 1978 (D. Schlessinger, editor), ASM Publications, 1977 (in press).